Electro-transformation of Lactobacillus spp-PDF

Overview

While there is a wide variety of methods used for the electrotransformation of Lactobacillus spp., they all share a typical format.

• Dilution: This determines the amount of overnight culture to add to new growth media
• Growth: These are the conditions used to grow the cells on the day you plan to transform.
• Washing: Cells are repeatedly centrifuged and resuspended in a solution of some type.
• Buffer: This is what is used for the final resuspension and electroporation.
• Concentration: This determines how much Electroporation buffer to add relative to the volume of the growth media.
• Voltage: How hard to shock em.
• Recovery: What to do with your cells after the shock.

This page will list many of these protocols in a standard format so that you can compare them and choose the one that works for you. In some cases an apples to apples comparison may not be acceptable (e.g. the differences in electroporators can be dramatic) so be sure to check the actual paper.

Procedures

Josson (1989)

• Species = L. plantarum, L. casei.
• Dilution = 1/50
• Growth = MRS + 20mM DL threonine, 37°C, OD₆₀₀=0.5-1.0
• Wash = 2x w/ water (RT°C)
• Buffer = 30% PEG 1000
• Concentration = 10X cell pellet volume
• Voltage = 8,500 V/cm
• Recovery = 30min ice / 2hrs MRS

Bringel (1990)

• Species = L. plantarum
• Dilution = to OD₆₀₀=0.05
• Growth = MRS + 1% Glycine, 0.75M Sorbitol, 30°C, Shake vigorously, OD₆₀₀=0.3 (~4 hours)
• Wash = 3x w/ water (RT°C)
• Buffer = 30% PEG
• Concentration = 1/125
• Voltage = 12,500 V/cm
• Recovery = 30min ice / 3hrs MRS

Posno (1991)

• Species = L. casei, L. pentosus, L. plantarum, L. acidophilus, L. fermentum, and L. brevis
• Dilution = 1/50
• Growth = MRS + 1% Glycine, 37°C, 3-4 hours
• Wash = 5mM NaH₂PO₄ 1mM MgCl₂ (Ice Cold)
• Buffer = 0.3M Sucrose 5mM NaH₂PO₄ 1mM MgCl₂ (Ice Cold)
• Concentration = 1/100
• Voltage = 7,000 V/cm
• Recovery = 90min MRS

Aukrust (1992)

• Species = L. plantarum, L. sake
• Dilution = 1/50
• Growth = 1% Glycine, 30°C, OD₆₀₀.=0.6
• Wash = 1mM MgCl₂ (RT°C)
• Buffer = 30% PEG 1500
• Concentration = 1/100
• Voltage = 7,500 V/cm
• Recovery = 2hrs MRS 0.5M Sucrose, 0.1M MgCl₂

Wei (1995)

• Species = L. fermentum
• Dilution = 1/50
• Growth = 1% Glycine, 37°C
• Wash = 5mM NaH₂PO₄ 1mM MgCl₂ (0°C)
• Buffer = 0.9M Sucrose 3mM MgCl₂
• Concentration = 1/100
• Voltage = 12,500 V/cm
• Recovery = 2hrs MRS 0.5M Sucrose, 0.1M MgCl2

Berthier (1996)

• Species = Lb. sake
• Dilution = unspecified
• Growth = MRS, 30°C
• Wash = 10mM MgCl₂ (0°C)
• Buffer = 0.5M Sucrose, 10%Glycerol
• Concentration = 1/200
• Voltage = 7,000 V/cm
• Recovery = 2hrs MRS, 80mM MgCl₂

Thompson (1996)

• Species = Lb. plantarum
• Dilution = 1/20
• Growth = MRS, 6% Glycine, 37°C
• Wash = 2X water, 1X 50mM EDTA, 2X 0.3M Sucrose ALL ICE COLD
• Buffer = 0.3M Sucrose
• Concentration = 1/50
• Voltage = 7,500 V/cm
• Recovery = 2hrs MRS

Serror (2002)

• Species = L. delbrueckii
• Dilution = Unpecified
• Growth = MRS, 42°C to OD₆₀₀ = 1.7
• Wash = 3X ice-cold EB buffer (1 mM MgCl₂, 5mM KH₂PO₄)
• Buffer = 0.4M Sucrose, 1mM MgCl₂, 5mM KH₂PO₄; PH: 6)
• Concentration = 1/30
• Heat-Shock = 45°C for 20 mins, then ice for 10 mins
• Voltage = 5,000V/cm
• Recovery = 3hrs Milk Medium ((0.2 M sucrose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25 mM MgCl₂) 37°C

Alegre (2004)

• Species = L. plantarum
• Dilution = 1/10
• Growth = LAB, 37°C, MRS, 30°C
• Wash = 2X 10mL 10 mM chilled MgCl₂, 1X 10mL chilled 0.5M Sucrose, 10% Glycerol
• Buffer = 0.5M Sucrose, 10% Glycerol
• Concentration = 1/30
• Voltage = 13,000 V/cm
• Recovery = 2hrs MRS, 80mM MgCl2

Kim (2005)

• Species = L. acidophilus, L. helveticus, L. brevis
• Dilution = 1/50
• Growth = 1% Glycine, 37°C, OD₆₀₀ = 0.2-0.3
• Wash = Ice 10mins, 2x w/ 5mM NaH₂PO₄ 1mM MgCl₂ (0°C)
• Buffer = 1M Sucrose 3mM MgCl₂
• Concentration = 1/100
• Voltage = 12,500 V/cm
• Recovery = 2hrs MRS 0.5M Sucrose, 0.1M MgCl2

Mason (2005)

• Species = Lb. casei, Lb. crispatus, Lb. delbreuckii, Lb. plantarum, Lb. salivarius
• Dilution = 1/6
• Growth = MRS, 8% Glycine, 90mins, 37°C
• Wash = 2X water; 1X 50mM EDTA; 2X 0.3M Sucrose. ALL ICE COLD
• Buffer = 0.3M Sucrose
• Concentration = 1/120
• Voltage = 7,500 V/cm
• Recovery = 2hrs MRS

Speer (2012)

• Species = L. plantarum
• Dilution = 1/50
• Growth = 2% Glycine, MRS, 3 hrs, 37°C, shaking
• Wash = 2X water; 1X 50mM EDTA; 2X E Buffer. ALL ICE COLD
• Buffer = 0.5M Sucrose, 10%Glycerol
• Concentration = 1/100
• Voltage = 12,000 V/cm
• Recovery = 2hrs MRS 30°C

Notes

1. Centrifugation at 4000 rpm for 2 minutes was sufficient to pellet the competent cells to give a clear supernatant.
2. Most of these papers use the Bio-Rad Gene Pulser and have time constants in the range of 9-10 ms with the lower voltages (7-9kV/cm). If you’re using a preset electroporator (like the Eppendorf 2510) you should try a higher voltage (~1200) as your time constand will be in thee range of 5-6 ms. If you’re using PEG in your electroporation buffer expect your time constant to be significantly lower.
3. If you get ZERO TRANSFORMANTS after trying multiple methods, chances are that your plasmid is incompatible with the strain you’re transforming. This page exists because this happened to me; and after I had tried ALL the protocols listed here, I got a new plasmid and it worked beautifully — Mike.

References

• Alegre, M.T, Rodrıguez, M.C., and J.M. Mesas. (2004) “Transformation of Lactobacillus plantarum by electroporation with in vitro modified plasmid DNA.” FEMS Microbiology Letters 241: 73-77)

• Aukrust, T. and H. Blom (1992). “Transformation of Lactobacillus strains used in meat and vegetable fermentaions.” Food Research International 25(4): 253-261.

• Berthier, F., M. Zagorec, et al. (1996). “Efficient transformation of Lactobacillus sake by electroporation.” Microbiology-Uk 142: 1273-1279.

• Bringel, F. and J.C. Hubert. (1990) “Optimized transformation by electroporation of Lactobacillus plantarum strains with plasmid vectors” Applied Microbiology and Biotechnology 33:664-670

• Josson, K., T. Scheirlinck, et al. (1989). “Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii.” Plasmid 21(1): 9-20.

• Kim, Y.H., Han, K.S., Oh, s., You, S. and S.H. Kim (2005) “Optimization of technical conditions for the transformation of Lactobacillus acidophilus strains by electroporation.” Journal of Applied Microbiology 99: 167–174

• Mason, C. K., M. A. Collins, et al. (2005). “Modified electroporation protocol for Lactobacilli isolated frorn the chicken crop facilitates transformation and the use of a genetic tool.” Journal of Microbiological Methods 60(3): 353-363.

• Posno, M., R. J. Leer, et al. (1991). “Incompatibility of Lactobacillus vectors with replicons derived from small cryptic plasmids and segregational instability of the introduced vectors.” Applied and Environmental Microbiology 57(6): 1822-1828.

• Serror, P., Sasaki, T., Ehrlich, S.D. and Emmanuelle Maguin. (2002) “Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids.” Applied and Environmental Microbiology, p.46–52

• Speer, M.A. and T.L. Richard (2012) Will Soon Exist.

• Thompson, K. and M. A. Collins (1996). “Improvement in electroporation efficiency for Lactobacillus plantarum by the inclusion of high concentrations of glycine in the growth medium.” Journal of Microbiological Methods 26(1-2): 73-79.

• Wei, M. Q., C. M. Rush, et al. (1995). “An improved method for the transformation of Lactobacillus strains using electroporation.” Journal of Microbiological Methods 21(1): 97-109.